Automated system for two-dimensional electrophoresis

ABSTRACT

The present invention provides an integrated, fully automated, high-throughput system for two-dimensional electrophoresis comprised of gel-making machines, gel processing machines, gel compositions and geometries, gel handling systems, sample preparation systems, software and methods. The system is capable of continuous operation at high-throughput to allow construction of large quantitative data sets.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 09/339,164, filed Jun. 24, 1999, issued Jun. 12, 2001 as U.S.Pat. No. 6,245,206 which was a division of U.S. patent application Ser.No. 08/881,761, filed Jun. 24, 1997, that issued Nov. 30, 1999 as U.S.Pat. No. 5,993,627.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States Government support undercooperative agreement number 70NANB5H1075 awarded by the NationalInstitute of Standards and Technology.

BACKGROUND OF THE INVENTION

The invention relates to the field of electrophoretic separations ofmacromolecules and in particular, to the automation of two-dimensionalelectrophoretic separations used in the analysis of proteins. Suchtwo-dimensional procedures typically involve sequential separations byisoelectric focusing (IEF) and SDS slab gel electrophoresis, and anautomated 2-D method thus involves manufacture and use of gel media forboth isoelectric focusing and SDS electrophoresis, together with meansfor protein detection and quantitation. Two-dimensional electrophoresistechnology forms the basis of the expanding field of proteomics, andhence automation of the procedure is a critical requirement for scale-upof efforts to build proteome databases comprising all the proteins ofcomplex organisms such as man. To date no successful automation effortshave been reported, despite the use of bench-scale 2-D electrophoresisin more than 5,000 scientific publications.

The publications and other materials used herein to illuminate thebackground of the invention and in particular, cases to provideadditional details respecting the practice, are incorporated herein byreference, and for convenience are referenced in the following text andrespectively grouped in the appended List of References. Elements of theinvention are disclosed in our Disclosure Documents 393753, 393754 and412899.

Isoelectric Focusing (IEF)

A protein is a macromolecule composed of a chain of amino acids. Of the20 amino acids found in typical proteins, four (aspartic and glutamicacids, cysteine and tyrosine) carry a negative charge and three (lysine,arginine and histidine) a positive charge, in some pH range. A specificprotein, defined by its specific sequence of amino acids, is thus likelyto incorporate a number of charged groups along its length. Themagnitude of the charge contributed by each amino acid is governed bythe prevailing pH of the surrounding solution, and can vary from aminimum of 0 to a maximum of 1 charge (positive or negative depending onthe amino acid), according to a titration curve relating charge and pHaccording to the pK of the amino acid in question. Under denaturingconditions in which all of the amino acids are exposed, the total chargeof the protein molecule is given approximately by the sum of the chargesof its component amino acids, all at the prevailing solution pH.

Two proteins having different ratios of charged, or titrating, aminoacids can be separated by virtue of their different net charges at somepH. Under the influence of an applied electric field, a more highlycharged protein will move faster than a less highly charged protein ofsimilar size and shape. If the proteins are made to move from a samplezone through a non-convecting medium (typically a gel such aspolyacrylamide), an electrophoretic separation will result.

If, in the course of migrating under an applied electric field, aprotein enters a region whose pH has that value at which the protein'snet charge is zero (the isoelectric pH), it will cease to migraterelative to the medium. Further, if the migration occurs through amonotonic pH gradient, the protein will “focus” at this isoelectric pHvalue. If it moves toward more acidic pH values, the protein will becomemore positively charged, and a properly-oriented electric field willpropel the protein back towards the isoelectric point. Likewise, if theprotein moves towards more basic pH values, it will become morenegatively charged, and the same field will push it back toward theisoelectric point. This separation process, called isoelectric focusing,can resolve two proteins differing by less than a single charged aminoacid among hundreds in the respective sequences.

A key requirement for an isoelectric focusing procedure is the formationof an appropriate spatial pH gradient. This can be achieved eitherdynamically, by including a heterogeneous mixture of charged molecules(ampholytes) into an initially homogeneous separation medium, orstatically, by incorporating a spatial gradient of titrating groups intothe gel matrix through which the migration will occur. The formerrepresents classical ampholyte-based isoelectric focusing, and thelatter the more recently developed immobilized pH gradient (IPG)isoelectric focusing technique. The IPG approach has the advantage thatthe pH gradient is fixed in the gel, while the ampholyte-based approachis susceptible to positional drift as the ampholyte molecules move inthe applied electric field. The best current methodology combines thetwo approaches to provide a system where the pH gradient is spatiallyfixed but small amounts of ampholytes are present to decrease theadsorption of proteins onto the charged gel matrix of the IPG.

It is current practice to create IPG gels in a thin planar configurationbonded to an inert substrate, typically a sheet of Mylar plastic whichhas been treated so as to chemically bond to an acrylamide gel (e.g.,Gelbond® PAG film, FMC Corporation). The IPG gel is typically formed asa rectangular plate 0.5 mm thick, 10 to 30 cm long (in the direction ofseparation) and about 10 cm wide. Multiple samples can be applied tosuch a gel in parallel lanes, with the attendant problem of diffusion ofproteins between lanes producing cross contamination. In the case whereit is important that all applied protein in a given lane is recovered inthat lane (as is typically the case in 2-D electrophoresis), it hasproven necessary to split the gel into narrow strips (typically 3 mmwide), each of which can then be run as a separate gel. Since theprotein of a sample is then confined to the volume of the gelrepresented by the single strip, it will all be recovered in that strip.Such strips (Immobiline DryStrips) are produced commercially byPharmacia Biotech.

While the narrow strip format solves the problem of containing sampleswithin a recoverable, non-cross-contaminating region, there remainsubstantial problems associated with the introduction of sample proteinsinto the gel. Since protein-containing samples are typically prepared ina liquid form, the proteins they contain must migrate, under theinfluence of the electric field, from a liquid-holding region into theIPG gel in order to undergo separation. This is typically achieved bylightly pressing an open-bottomed rectangular frame against the planargel surface so that the gel forms the bottom of an open-topped butotherwise liquid-tight vessel (the sample well). The sample is thendeposited in this well in contact with the gel surface forming thebottom of the well. Since all of the sample protein must pass through asmall area on the surface of the gel (the well bottom) in order to reachthe gel interior, the local concentration of protein at the entry pointcan become very high, leading to protein precipitation. The sample entryarea is typically smaller than the gel surface forming the well bottombecause the protein migrates into the gel under the influence of anelectric field which directs most of it to one edge of the well bottom,tending to produce protein precipitation. The major source ofprecipitation, however, is provided by the charged groups introducedinto the gel matrix to form the pH gradient in IPG gels: these groupscan interact with charges on the proteins (most of which are not attheir isoelectric points at the position of the application point andhence have non-zero net charges) to bind precipitates to the gel. It iscommon experience that separations of the same protein mixture on aseries of apparently identical IPG gels can yield very differentquantitative recoveries of different proteins at their respectiveisoelectric points, indicating that the precipitation phenomenon mayvary from gel to gel in unpredictable ways, thereby frustrating thegeneral use of IPG gels for quantitative protein separations.

Recently, methods have been introduced in which the IPG strip isre-swollen, from the dry state, in a solution containing sampleproteins, with the intention that the sample proteins completelypermeate the gel at the start of the run.

Isoelectric focusing separation of proteins in an immobilized pHgradient (IPG) is extensively described in the art. The concept of theIPG is disclosed in U.S. Pat. No. 4,130,470 and is further described innumerous publications. The IPG gel strips manufactured are generally ofsimple planar shape.

A series of disclosures have dealt with various configurations ofcavities (“sample wells”)used for the application ofmacromolecular-containing samples to the surfaces of gels, mostfrequently slab gels used for protein or nucleic acid separations. Ineach case, these sample wells were designed to concentratemacromolecules in the sample into a thin starting zone prior to theirmigration through the resolving gel. The following references describethe use of devices placed against a gel to form wells: U.S. Pat. No.5,304,292 describes the use of pieces of compressible gasket to formwell walls at the top of a slab where the ends of the pieces touch thetop edge of the slab. U.S. Pat. No. 5,164,065 describes a shark's toothcomb used in combination with DNA sequencing gels.

Several references describe automated devices for creating gradients ofpolymerizable monomers. Such systems have been used for making porositygradient gels used in molecular weight separations of proteins. Altlandet al. (Altland, K. and Altland, A. Pouring reproducible gradients ingels under computer control, Clin.Chem. 30(12 Pt 1): 2098-2103, 1984)shows the use of such systems for creating the gradients of titratablemonomers used in the creation of IPG gels. U.S. Pat. No. 4,169,036describes a system for loading slab-gel holders for electrophoresisseparation. U.S. Pat. No. 4,594,064 discloses an automated apparatus forproducing gradient gels. Hence, use of a computer-controlled gradientmaker in manufacturing IPG and other gels is known in the art.

One alternative method of running IPG strips in an IsomorpH device isdisclosed in Disclosure Document No. 342751 (Anderson, N. L., entitled“Vertical Method for Running IPG Gel Strips”). The disclosed device usessample wells pressed against the gel surface, but otherwise completelyclosed, so that the assembly could be rotated into a verticalorientation, thus allowing closer packing of gels and a greater gelcapacity in a small instrument footprint. Additional methods aredisclosed in Disclosure Document No's. 393753 (Anderson, N. L., Goodman,Jack, and Anderson, N. G., entitled “Gel Strips for ProteinSeparation”)and 412899 (Anderson, N. L., Goodman, Jack, and Anderson, N.G., entitled “Automated System for Two-Dimensional Electrophoresis”).

Systems for making non-planar slab gels are also known in the art andare disclosed in the following references: U.S. Pat. No. 5,074,981discloses a substitute for submarine gels using an agarose block that isthicker at the ends and hangs into buffer reservoirs. U.S. Pat. No.5,275,710 discloses lane-shaped gels formed in a plate and gel-filledholes extending down from the plate into buffer reservoirs. These gelsystems, however, do not provide a gel which can be given across-section that is optimal for producing high-resolution proteinseparation. Furthermore, these systems cannot incorporate varyingcross-sections along the length of a gel as required.

SDS Slab Gel Electrophoresis

Charged detergents such as sodium dodecyl sulfate (SDS) can bindstrongly to protein molecules and “unfold” them into semi-rigid rodswhose lengths are proportional to the length of the polypeptide chain,and hence approximately proportional to molecular weight. A proteincomplexed with such a detergent is itself highly charged (because of thecharges of the bound detergent molecules), and this charge causes theprotein-detergent complex to move in an applied electric field.Furthermore, the total charge also is approximately proportional tomolecular weight (since the detergent's charge vastly exceeds theprotein's own intrinsic charge), and hence the charge per unit length ofa protein-SDS complex is essentially independent of molecular weight.This feature gives protein-SDS complexes essentially equalelectrophoretic mobility in a non-restrictive medium. If the migrationoccurs in a sieving medium, such as a polyacrylamide gel, however, large(long) molecules will be retarded compared to small (short) molecules,and a separation based approximately on molecular weight will beachieved. This is the principle of SDS electrophoresis as appliedcommonly to the analytical separation of proteins.

An important application of SDS electrophoresis involves the use of aslab-shaped electrophoresis gel as the second dimension of atwo-dimensional procedure. The gel strip or cylinder in which theprotein sample has been resolved by isoelectric focusing is placed alongthe slab gel edge and the molecules it contains are separated in theslab, perpendicular to the prior separation, to yield a two-dimensional(2-D) separation. Fortunately, the two parameters on which this 2-Dseparation is based, namely isoelectric point and mass, are almostcompletely unrelated. This means that the theoretical resolution of the2-D system is the product of the resolutions of each of the constituentmethods, which is in the range of 150 molecular species for both IEF andSDS electrophoresis. This gives a theoretical resolution for thecomplete system of 22,500 proteins, which accounts for the intenseinterest in this method. In practice, as many as 5,000 proteins havebeen resolved experimentally. The present invention is aimed primarilyat the 2-D application, and includes means for automating the seconddimension SDS separation of a 2-D process to afford higher throughput,resolution and speed.

It is current practice to mold electrophoresis slab gels between twoflat glass plates, and then to load the sample and run the slab gelstill between the same glass plates. The gel is molded by introducing adissolved mixture of polymerizable monomers, catalyst and initiator intothe cavity defined by the plates and spacers or gaskets sealing threesides. Polymerization of the monomers then produces the desired gelmedia. This process is typically carried out in a laboratory setting, inwhich a single individual prepares, loads and runs the gel. A gasket orform comprising the bottom of the molding cavity is removed after gelpolymerization in order to allow current to pass through two oppositeedges of the gel slab: one of these edges represents the open (top)surface of the gel cavity, and the other is formed against its removablebottom. Typically, the gel is removed from the cassette defined by theglass plates after the electrophoresis separation has taken place, forthe purposes of staining, autoradiography, etc., required for detectionof resolved macromolecules such as proteins.

The concentrations of polyacrylamide gels used in electrophoresis arestated generally in terms of % T (the total percentage of acrylamide inthe gel by weight) and % C (the proportion of the total acrylamide thatis accounted for by the crosslinker used). N,N′-methylenebisacrylamide(“bis”)is typically used as crosslinker. Typical gels used to resolveproteins range from 8% T to 24% T, a single gel often incorporating agradient in order to resolve a broad range of protein molecular masses.

In most conventional systems used for SDS electrophoresis, use is madeof the stacking phenomenon first employed in this context by Laemmli, U.K. (1970) Nature 227, 680. In a stacking system, an additional gel phaseof high porosity is interposed between the separating gel and thesample. The two gels initially contain a different mobile ion from theion source (typically a liquid buffer reservoir) above them: the gelscontain chloride (a high mobility ion) and the buffer reservoir containsglycine (a lower mobility ion, whose mobility is pH dependent). Allphases contain Tris as the low-mobility, pH determining other buffercomponent and positive counter-ion. Negatively charged protein-SDScomplexes present in the sample are electrophoresed first through thestacking gel at its pH of approximately 6.8, where the complexes havethe same mobility as the boundary between the leading (Cl—) and trailing(glycine-) ions. The proteins are thus stacked into a very thin zone“sandwiched” between Cl— and glycine- zones. As this stacking boundaryreaches the top of the separating gel the proteins become unstackedbecause, at the higher separating gel pH (8.6), the protein-SDScomplexes have a lower mobility. Thus, in the separating gel, theproteins fall behind the stacking front and are separated from oneanother according to size as they migrate through the sievingenvironment of the lower porosity (higher % T acrylamide) separatinggel. In this environment, proteins are resolved on the basis of mass.

Pre-made slab gels have been available commercially for many years(e.g., from Integrated Separation Systems). These gels are prepared inglass cassettes much as would be made in the user's laboratory, andshipped from a factory to the user. More recently, methods have beendevised for manufacture of both slab gels in plastic cassettes (therebydecreasing the weight and fragility of the cassettes) and slab gelsbonded to a plastic backing (e.g., bonded to a Gelbond® Mylar® sheet orto a suitably derivatized glass plate). To date, allcommercially-prepared gels are either enclosed in a cassette or bondedto a plastic sheet on one surface (the other being covered by aremovable plastic membrane). Furthermore, all commercially-prepared gelshave a planar geometry.

Current practice in running slab gels involves one of two methods. A gelin a cassette is typically mounted on a suitable electrophoresisapparatus, so that one edge of the gel contacts a first buffer reservoircontaining an electrode (typically a platinum wire) and the opposite geledge contacts a second reservoir with a second electrode, steps beingtaken so that the current passing between the electrodes is confined torun mainly or exclusively through the gel. Such apparatus may be“vertical” in that the gel's upper edge is in contact with an upperbuffer reservoir and the lower edge is in contact with a lowerreservoir, or the gel may be rotated 90° about an axis perpendicular toits plane, so that the gel runs horizontally between a left and rightbuffer reservoir, as is disclosed in U.S. Pat. No. 4,088,561 (e.g.,“DALT” electrophoresis tank). Various configurations have been devisedin order to make these connections electrically, and to simultaneouslyprevent liquid leakage from one reservoir to the other (around the gel).

When used as part of a typical 2-D procedure, an IEF gel is appliedalong one exposed edge of such a slab gel and the proteins it containsmigrate into the gel under the influence of an applied electric field.The IEF gel may be equilibrated with solutions containing SDS, bufferand thiol reducing agents prior to placement on the SDS gel, in order toensure that the proteins the IEF gel contains are prepared to beginmigrating under optimal conditions, or else this equilibration may beperformed in situ by surrounding the gel with a solution or gelcontaining these components after it has been placed in position alongthe slab's edge.

A slab gel affixed to a Gelbond® sheet is typically run in a horizontalposition, lying flat on a horizontal cooling plate with the Gelbond®sheet down and the unbonded surface up. Electrode wicks communicatingwith liquid buffer reservoirs, or bars of buffer-containing gel, areplaced on opposite edges of the slab to make electrical connections forthe run, and samples are generally applied onto the top surface of theslab (as in the instructions for the Pharmacia ExcelGels).

It is current practice to detect proteins in 2-D gels either by stainingthe gels or by exposing the gels to a radiosensitive film or plate (inthe case of radioactively labeled proteins).

Staining methods include dye-binding (e.g., Coomassie Brilliant Blue),silver stains (in which silver grains are formed in protein-containingzones), negative stains in which, for example, SDS is precipitated by Znions in regions where protein is absent, or the proteins may befluorescently labeled. In each case, images of separated protein spotpatterns can be acquired by scanners, and this data reduced to providepositional and quantitative information on sample protein compositionthrough the action of suitable computer software.

Additional methods are disclosed in Disclosure Document No's. 393754(Anderson, N. L., Goodman, Jack, and Anderson, N. G., entitled“Apparatus and Methods for Casting and Running Electrophoresis SlabGels”) and 412899 (Anderson, N. L., Goodman, Jack, and Anderson, N. G.,entitled “Automated System for Two-Dimensional Electrophoresis”).

Sample Preparation

Protein samples to be analyzed using 2-D electrophoresis are typicallysolubilized in an aqueous, denaturing solution such as 9M urea, 2% NP-40(a non-ionic detergent), 2% of a pH 8-10.5 ampholyte mixture and 1%dithiothreitol (DTT). The urea and NP-40 serve to dissociate complexesof proteins with other proteins and with DNA, RNA, etc. The amrpholytemixture serves to establish a high pH (˜9) outside the range where mostproteolytic enzymes are active, thus preventing modification of thesample proteins by such enzymes in the sample, and also complexes withDNA present in the nuclei of sample cells, allowing DNA-binding proteinsto be released while preventing the DNA from swelling into a viscous gelthat interferes with IEF separation. The purpose of the DTT is to reducedisulfide bonds present in the sample proteins, thus allowing them to beunfolded and assume an open structure optimal for separation bydenaturing IEF. Samples of tissues, for example, are solubilized byrapid homogenization in the solubilizing solution, after which thesample is centrifuged to pellet insoluble material and DNA, and thesupernatant collected for application to the EEF gel.

Because of the likelihood that protein cysteine residues will be comeoxidized to cysteic acid or recombine and thus stabilize refolded, notfully denatured protein structures during the run, it is desirable tochemically derivatize the cysteines before analysis. This is typicallyaccomplished by alkylation to yield a less reactive cysteine derivative.

Use of 2-D Electrophoresis

Two-dimensional electrophoresis is widely used to separate from hundredsto thousands of proteins in a single analysis, in order to visualize andquantitate the protein composition of biological samples such as bloodplasma, tissues, cultured cells, etc. The technique was introduced in1975 by O'Farrell, and has been used since then in various forms in manylaboratories.

The gel systems known in the art or referenced above, however, do notprovide an integrated, fully automated, high-throughput system fortwo-dimensional electrophoresis of proteins. Moreover, current IPG andslab gel systems are not fully automated, wherein all operationsincluding gel casting, processing, sample loading, running and finaldisposition are carried out by an integrated, fully automated system.Current gel systems cannot be fully controlled by a computer and cannotsystematically vary gel, process, sample load and run parameters,provide positive sample identification, and cannot collect process datawith the object of optimizing the reproducibility and resolution of theprotein separations.

OBJECT OF THE INVENTION

It is an object of the present invention to provide means for fullyautomated, high-throughput two-dimensional electrophoresis of proteins.

It is a further object of the present invention to provide a means ofalkylating protein sulfhydryl groups in an automated manner.

It is a further object of the present invention to provide an IPG gelsystem optimized for use in a two-dimensional gel system wherein alloperations including gel casting, processing, sample loading, runningand final disposition (either by staining for protein detection orapplication to a second dimension slab gel for use in a two-dimensionalprotein separation) are carried out by an automated system.

It is a further object of the present invention to provide an IPG gelwhich is not restricted to a planar geometry, but which instead can begiven any cross-section judged optimal for producing a high-resolutionprotein separation, and can incorporate varying cross-sections along itslength as required.

It is a further object of the present invention to provide an IPG gelstrip system that can be fully controlled by a computer, therebyaffording the opportunity to systematically vary gel, process, sampleload and run parameters and collect process data with the object ofoptimizing the reproducibility and resolution of the separation.

It is a further object of the present invention to provide a system forSDS slab gel electrophoresis offering facile automation (the slab gelsshould be easily handled in a robotic manner during casting, loading andrunning).

It is a further object of the present invention to provide accurateplacement of the sample with respect to the plane of the slab gel, so asto avoid migration of sample macromolecules in a distribution that isasymmetric with respect to the plane of the slab gel, i.e., along onesurface.

It is a further object of the present invention to provide effective andeven cooling of the slab gel surface so that voltage (and hence heatgenerated) can be increased, with attendant improvements in gelresolution (due to shorted run times, and consequently decreaseddiffusion time).

It is a further object of the invention to provide facile automation ofslab gel staining and scanning.

It is a further object of the invention to provide automated means forthe recovery of selected protein spots or gel zones for the purpose ofprotein identification and characterization by means such asmicrochemical sequencing or mass spectrometry.

SUMMARY OF THE INVENTION

The present invention provides an integrated, fully automated,high-throughput system for two-dimensional electrophoresis comprised ofgel-making machines, gel processing machines, gel compositions andgeometries, gel handling systems, sample preparation systems, softwareand methods. The system is capable of continuous operation athigh-throughput, to allow construction of large quantitative data sets.

Sample Preparation

Automated means are provided for treatment of protein-containing samplesto effect the reduction and alkylation of cysteine sulfhydryl groupscontained therein, with the object of preventing protein loss in the 2-Dprocess through protein aggregation or refolding associated withsulfhydryl re-oxidation during the run.

IEF

IPG gels are cast in a computer-controlled mold system capable ofrepeatedly casting a gel on a film support, advancing the support,cutting off the strip of support carrying the fresh gel, and presentingthe strip to a robotic arm. The robotic arm subsequently carries the IPGstrip and inserts it in a sequence of processing stations that implementsteps required to prepare the IPG and use it, including washing, drying,rehydration, sample loading, and subjection to high voltage.

The approach used in casting the IPG gel allows the shape of the gel todepart from conventional flat planar strip geometry. The method ofsample loading allows the sample to be applied over a large area of thegel. Such a gel format can provide. an improved two-stage separationsystem: a first stage in which the proteins are separated in aminimally-restrictive, ideally fluid medium by isoelectric focusing in achannel or surface layer containing conventional ampholytes butsurrounded by an IPG gel that establishes the pH gradient, andcontinuing on to a second stage in which the proteins are imbibed by thesurrounding IPG gel at, or near their isoelectric points and maintainedin stable, focused positions until the end of the run.

SDS Slabs

SDS slab gels used for the second dimension separation are formed in anautomated mold which plays the role of the gel-forming cassette of aconventional system. By using an approach analogous to injectionmolding, the gel is no longer required to assume a homogeneous planarconfiguration. In effect, a three-phase gel may be constructed, havingregions corresponding to the separating gel, stacking gel and upperbuffer reservoirs of a conventional slab gel system. Polymerizable gelsolutions can be fed to the mold by one or more computer-controlledpumping devices, thus facilitating the creation of multiple zones of gelhaving different electrochemical properties. An upper electrode in theform of a rigid bar is polymerized into one region of the slab gel,allowing it to be manipulated and transported “bare” (i.e., without anysurface protection or coating) by a second robotic arm (i.e., nocassette).

A slot or other means is provided for introducing a sample (usually inthe form of a first dimension gel rod or strip) into or onto the slab.The slab is “run” (voltage applied) while it is hanging in a bath ofcooled, circulating insulating liquid, such as silicone oil. The oilprevents evaporation of water from the planar gel surfaces as the gelruns (a function typically performed by the glass plates of aconventional gel cassette) and prevents joule heat caused by theelectrophoresis current from raising the temperature of the gelappreciably. The gel contacts a layer of aqueous solvent underlying theoil, serving as a lower buffer (with suitable electrodes). The lowdensity of the oil keeps it above and unmixed with the lower aqueousbuffer.

After the run, the slab gel is carried by the second robotic arm to asuccession of tanks containing a series of solutions needed to effectstaining of the protein spots or bands on the gel. Because ofdifferences in the physical densities of these solutions, the stainingcan make use of the fact that, as solutes are exchanged between thehanging gel slab and the solution, a lamina forms at the surfaces of theslab gel that has a density different from that of the bulk solvent.Because of this difference, the fluid in this lamina either rises orfalls as a curtain along the slab surface, and is replaced by freshsolvent. Hence, depleted solution accumulates at either the top orbottom of the tank, where it can be removed and replaced with freshsolution. After staining, the gel can be transported by the robotic armto a scanner where it can be digitized for computer analysis.

Software

The entire process can be controlled by a computer running software thatboth drives the creation and processing of each gel and collects processdata from sensors placed at strategic points in the production line soas to allow quality control and optimization. A scheduling algorithm isimplemented in software so that each sample can be run with differentgel parameters, if desired, while ensuring that the manifold actionsrequired to process one sample do not interfere with actions required toprocess other gels in the system (e.g., so that the arm used totransport IPG gels between processing stations is not required to be intwo places at once).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the entire automated 2-Delectrophoresis process.

FIG. 2 illustrates sample preparation using a size exclusion column.

FIG. 3 illustrates an IPG gradient maker and mold system.

FIG. 4 is a schematic cross-section through an IPG mold system.

FIG. 5 shows a series of six alternative cross-sections for IPG gelsformed by various mold activities.

FIG. 6 is a schematic view of an IPG strip in a horizontal position withthe gel-side on top of a base plate in position for sample loading.

FIG. 7 is a side view of an IPG carrier arm and an IPG slot run.

FIG. 8 illustrates the sequence of actions of a slab gel mold duringcasting operation.

FIG. 9 illustrates alternative forms of slab gels.

FIG. 10 is an end view of slab gel run tanks.

FIG. 11 is an end view of slab gel staining tanks with the slab carrierarm, and gel carriers.

FIG. 12 illustrates the placement of a slab gel on a scanning platformby a slab carrier arm and configuration of fluorescence illumination.

FIG. 13 illustrates the sequence of actions of a spot excision punch.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The preferred embodiment of the automated system for 2-D electrophoresisdescribed is a continuously-operating production line, making each gel(both IEF and SDS) just as needed, and capable of undertaking all stepsof the process (FIG. 1)—from loading of sample onto the first dimensiongel to final entry of protein quantitation data into a computerdatabase.

Sample Preparation

In order that proteins retain constant chemical properties during theprocess of separation by IPG-IEF and SDS electrophoresis, it isimportant that the sulfhydryl (SH) groups of the cysteine residues thatthey contain not be allowed to reform disulfide bridges or becomeoxidized to cysteic acid during the separation process. In the preferredembodiment, the protein cysteine residues are permanently renderedstable by alkylation with iodoacetamide or one of its uncharged orzwitterionic derivatives (such as S+2-amino-5-iodoacetamido-pentanoicacid), which introduces a very hydrophilic group at every cysteineposition but does not change the protein's net charge or apparentisoelectric point and has a negligible effect on protein mass. Thisderivatization is implemented in an automated fashion using a sizeexclusion gel filtration column to exchange the proteins out of theinitial sample solubilization solution, through a reagent zonecontaining alkylating reagent, and finally into a medium suitable forapplication to an IPG gel. The size exclusion media is chosen so as toexclude proteins but not low molecular weight solvents (e.g.,polyacrylamide beads such as BioRad P-6 BioGel). In practice, a samplecontaining a sulfhydryl reducing reagent such as DTT is removed from avial selected by a conventional autosampler such as is used in highperformance liquid chromatography (HPLC), directed by a valve at thehead of the column onto a column which has been pre-equilibrated withthe final sample solvent and a zone (immediately preceding the sample)containing alkylating reagent in sufficient excess to ensure rapidreaction with protein cysteines. Once the sample zone is loaded, thevalve switches to deliver a stream of final sample solvent that propelsall the zones down the column and prepares the column for the succeedingcycle. As the initial sample zone moves down the column the proteinmolecules, because of their greater size, fail to penetrate into theparticles of the column packing and hence move forward at a greaterspeed than that of the bulk solvent, which freely exchanges into thevolume of the porous particles. This principle of separation is wellknown in the art. The proteins thus move into the zone of alkylatingreactant, react there, and finally move even farther forward into thepreceding zone of final sample solvent. This procedure thus ensuresalkylation of protein sulfhydryls and removal of any low molecularweight contaminants as well. The sample is then ready for application toan IPG gel.

FIG. 2 illustrates a sample preparation apparatus which uses a sizeexclusion column. Diagram A depicts the arrangement of the components ofthe sample preparation apparatus. A size exclusion column 1 is connectedto one of a series of input liquid streams 2, 3 and 4 by amulti-position switching valve 5, with liquid flow into the columndriven by pump 6. Initially column 1 is equilibrated with liquid 4.Input 2 delivers the crude sample from a conventional autosampler orother device. Input 3 delivers a stream of reagent required to effect achemical treatment of the sample proteins (typically a sulfhydrylalkylating reagent), and input 4 delivers a stream of column eluent (thesolvent in which the sample proteins will ultimately emerge). Liquidemerging from the size exclusion column flows through a UV absorbance orother column monitor flow cell 7 and thereby to a multiport valve 8 thatdirects the eluent either to waste 9 or to a sample collection vessel10.

Diagrams B through H depict steps in the operation of the column toeffect sample protein derivatization. In diagram B, the column 1 isequilibrated with eluent through connection of its input to eluentreservoir 4 by input valve 5, and its output to waste 9 by output valve8. Pump 6 and UV monitor 7 are not shown for clarity: the pump isassumed to remain on during the sequence of operations, deliveringliquid continuously through the column. In C, a zone of alkylationreagent 11 is introduced onto the column by switching the input valve 5to draw solvent from the alkylation reagent reservoir 3. In diagram D, azone of sample is introduced after the alkylating reagent zone, saidsample zone comprising a solvent phase 12 and a protein solute phase 13.In diagram E, input to the column once again switches to eluent, pushingthe sample and alkylation zones down the column.

As the sample solvent zone moves down the column, the proteins itinitially contained are excluded from the matrix of the size exclusioncolumn and hence advance into the alkylation zone (a well known featureof such columns when used in desalting applications). During thisperiod, the proteins are exposed to the alkylating reagents and theircomponent sulfhydryl groups are alkylated to prevent re-folding of theproteins in subsequent stages of the 2D electrophoresis process. Indiagram F, the proteins in solute phase continue to advance down thecolumn faster than the proteins in solvent phase, and enter the leadingregion comprised of the first applied eluent phase. In diagram G, thealkylated proteins are collected by switching the output collectionvalve 8 to the sample collection position. In diagram H, continuing flowof eluent into the column forces the alkylation and initial samplesolvent phases out of the column in preparation for the column'sregeneration and re-use.

In an alternative embodiment, alkylation is performed with a negativelycharged reagent such as iodoacetic acid, thereby substituting a negativecharge at every alkylated protein sulfhydryl. When this reaction isaccomplished stoichiometrically, very basic proteins containing cysteineresidues are shifted towards more neutral isoelectric points, therebyfacilitating their detection on IEF gels.

IPG

The first operation of the 2D gel procedure is creation of anisoelectric focusing gel to effect the first dimension separation. Sucha separation is most effectively carried out in an immobilized pHgradient (IPG) gel, in which a gradient of polymerizable monomers isgelled to form a fixed spatial pH gradient.

Gradient

The compositional gradient required to form the desired pH gradient IPGgel can be produced by a system of four computer-controlled motorizedsyringes delivering, respectively, heavy gel monomer compositionformulated to yield a basic pH, light gel monomer composition formulatedto yield an acidic pH, a polymerization initiator such as ammoniumpersulfate, and a polymerization catalyst such as TEMED. A computerprogram constructed, for example, in the LabVIEW language, is used inconjunction with a computer and stepper motor control card (for example,a Compumotor AT6400 card) to produce a varying ratio between the speedof delivery of heavy and light components, while maintaining acontinuous delivery of initiator and catalyst required forpolymerization. Each of the four syringes is connected to a separatecomputer-controlled valve (e.g., a 6-port high pressure liquidchromatography valve in which each of two rotational positions connectsa fixed input with one of two lines and a fixed output with one of twoother lines) that allows connection of the syringe either to an externalreservoir, or to the delivery tubing system. When the syringe isconnected to the reservoir for refilling, the delivery system isconnected to a source of pressurized flush solvent (typically water)that displaces polymerizable monomer solutions from the delivery tubesto prevent blockage. In the delivery tubing system, the four componentflows emerging from the four valves are combined by appropriate tubingjunctions to yield one mixed fluid stream routed into the gradientdelivery tube in the mold.

An additional fifth syringe may be added to supply a third polymerizablemonomer solution of density and pH intermediate between the light andheavy monomer solutions, for the purpose of creating very wide pHgradients as a sequence of two two-component gradients (i.e., A→Bfollowed by B→C).

FIG. 3 schematically depicts the components of an IPG casting system. Avertically-oriented mold cavity is formed of a front mold half 14 and aback surface comprised of activated Gelbond® sheet 15. At each castingcycle, fresh Gelbond® is delivered to the mold from a roll 16 throughmotorized transport rollers 17. A small diameter rigid delivery tube 18extends into the mold from the top and may be raised out of the mold bylinear motion 19. A flexible tube 20 delivers a polymerizablecomposition to the delivery tube from a gradient maker having fivecomputer controlled syringes. Each syringe 21 is connected to the outputmanifold 22 through a 6-port valve 23 allowing the syringe to beconnected either to a liquid reservoir (e.g., liquid reservoir 24) forrefilling or to the output manifold. These syringes deliver one of threeacrylamide monomer solutions (24, 25, 26) ammonium persulfate 27 andTEMED 28. Valves attached to syringes drawing from reservoirs 24, 26, 27and 28 are shown in delivery position, while the valve attached to thesyringe drawing from reservoir 25 is shown in refill position.

Each syringe is driven by a motor 29 rotating a lead screw 30 thatgenerates linear motion of a block 31 attached to the syringe's plunger32. During the refilling of syringe 21 from its associated reservoir 24,the associated 6-port valve 23 connects the output manifold 22 to apressurized source of non-polymerizable solvent 33 (e.g., water), topurge the manifold and delivery tubes of polymerizable media (thisconfiguration shown for the middle syringe connected to reservoir 25).After delivering a gradient of polymerizable monomers to the mold, thedelivery tube 18 is raised by delivery tube motion 19 so that its openend lies in a block 34 through which air is sucked at high velocity byan air pump, from input 35 to output 36. A second linear motion 37carries a long straight pin 38 which can be inserted into the mold alongits axis or raised out of it.

The resulting compositional gradient must be delivered into a suitablemold such that a spatial gradient is maintained during gelation. Inorder to achieve this, the delivery tube delivering gel composition tothe mold is arranged on a vertical linear transport capable of insertingthe open end of the delivery-tube to the bottom of the vertical moldcavity, and raising it slowly as the gradient is dispensed so as todeposit successive elements of the gradient above one another (at therising meniscus of the liquid in the mold). When the gradient is thuscompleted, the delivery tube is raised fully out of the mold and into asuction block 34 mounted just above the gel mold. In this position,liquid emerging from the delivery-tube is sucked into a perpendicularwaste tube by the action of a vacuum, thereby providing a waste path forflush solvents directed through the delivery-tube between gradientdispensing operations in order to prevent blockage of the tube by anyremaining polymerizable components.

Suitable compositions for the four components combined to make an IPGare as follows. Solutions of polymerization catalyst and initiator(assuming that each comprises 10% of the total volume dispensed) are,respectively, 1.2% tetramethylethylenediamine (TEMED) and 1.2% ammoniumpersulfate (AP), both in water. The two solutions of polymerizablemonomers (whose proportions in the output stream vary to yield agradient of titratable monomers and physical density) may be made up asshown in the following Table to achieve a gradient over a range of pH 4to 9. The titratable monomers used are Immobilines® manufactured byPharmacia Biotech. Glycerol and deuterium oxide (heavy water) are usedto increase the density of one of the solutions, thus helping tostabilize the gradient formed in the mold through the interaction of theresulting density gradient and the earth's gravity.

TABLE 1 Heavy Light (pH 4) (pH 9) Immobiline pK 3.6 2,762 491microliters Immobiline pK 4.6 785 1,414 microliters Immobiline pK 6.2773 1,200 microliters Immobiline pK 7 75 988 microliters Immobiline pK8.5 834 236 microiiters Immobiline pK 9.3 738 2,209 microliters 31.8% T,5.6% C Acrylamide/bis in H₂O 0 9.83 ml 31.8% T, 5.6% C Acrylamide/bis inD₂O 9.83 0 ml Glycerol 6.25 0 ml D₂O (Heavy Water) 19.79 0 ml Water 025.46 ml 1 M Tris HCl, pH 7.0 8.17 8.17 ml Total 50.00 50.00 ml

Because the volume of the tubing connecting the gradient maker with themold is a significant fraction of the mold volume (even when narrow-boreHPLC tubing and connectors of inside diameter 0.010″ are used), it isnecessary to take account of this volume when dispensing a gradient.Hence, the procedure adopted and implemented in the control softwareconsists of five stages: 1) delivery of the first segment of the desiredgradient, equal in volume to the volume of the delivery tube, for thepurpose of replacing the flush solvent in the tube with polymerizablemonomer; 2) insertion of the delivery tube into the mold; 3) delivery ofthe remainder of the gradient while the delivery tube is raised(withdrawn from the mold) at a speed such that the delivered gradientcomposition is emitted at the rising surface of the liquid in the mold;4) following the gradient by a volume equal to the delivery tube volumeof the final “light” composition, for the purpose of forcing the sectionof the gradient remaining in the delivery tube into the mold while thedelivery tube continues to rise; and 5) removal of the delivery tubefrom the mold to the upper vacuum flush position where, followingswitching of the four valves, flush liquid is forced through thedelivery tube system to remove polymerizable material and to prepare thesystem for a subsequent gradient delivery.

Mold

In the preferred embodiment, the IPG is cast in a narrow vertical moldcavity formed by pressing a movable mold half against a sheet ofGelbond® PAG-activated plastic substrate which in turn is pressedagainst a fixed backing block whose temperature is controlled bycirculation of chilled or heated water through internal cavities. Thecavity in the movable mold half is surrounded on the sides and bottom byan O-ring groove with an O-ring to produce a liquid-tight seal againstthe Gelbond®. The Gelbond® substrate is made of Mylar® polyester plasticfilm treated in such a way as to produce on its active surface groups towhich an acrylamide gel can bond covalently, thus attaching the gel tothe Gelbond® substrate.

In the preferred embodiment, a longitudinal IPG gradient is formed inthe cavity by dispensing a varying composition of gelable monomers intothe cavity through a small diameter delivery tube. This delivery tuberises during the dispensing of the gradient, and consequent filling ofthe mold, so that the open end of the tube from which gelable monomeremerges is maintained at the rising level of the surface of the liquiddispensed into the mold. In addition, the gradient of gelable monomersis contrived so as to incorporate a physical density gradient thatevolves from heavy to light during the dispensing of the gradient. Sucha density gradient is produced by inclusion of a dense substance such asglycerol or deuterium oxide in place of a portion of the water presentin the “heavy” gradient component. A density gradient dispensed in the“heavy” to “light” sequence from a tube maintained at the rising surfaceof liquid in the mold gives rise to a stable composition gradient in themold which, when polymerized, yields an IPG.

FIG. 4 is a schematic cross-section of an IPG mold system viewed fromabove (i.e., looking down into the mold cavity 14 depicted in FIG. 3).In diagram A, the front IPG mold half 14 is pressed against the Gelbond®sheet 15 by a pneumatic cylinder 39, whose pressure bears on a fixedback plate 40. In thus example, the IPG mold cavity is of asemi-circular cross section 41. Lateral leakage of polymerizablecomponents is prevented by linear O-rings 42. Mold temperature can becontrolled by circulation of hot or cooled liquid through internalchannels of the fixed back plate. Polymerizable components areintroduced into the mold through moving delivery tube 18. A central holemay be formed in the IPG gel by polymerization with a pin 38 in placeinside the mold cavity.

In diagram B of FIG. 4, following extraction of the delivery tube andpin from the mold, pneumatic cylinder 39 retracts the front IPG moldhalf 14, and rollers 17 cause the Gelbond® support 15 to slide laterallyacross the face of fixed back plate 40, thereby ejecting the IPG gel 43on its Gelbond® substrate from the mold, in preparation for anothercycle. A rotary blade 44 cuts the Gelbond® by moving vertically alongthe mold, thereby releasing a strip of Gelbond® carrying thenewly-formed IPG gel. The gel produced has a longitudinal hole 45.

It is important to ensure that the gradient of the resulting gel reacheshydrostatic equilibrium (and hence proper gradient shape) beforepolymerization, and yet is fully polymerized (with completeincorporation of gelable monomers into the gel polymer matrix) beforeremoval from the mold. This result is achieved by increasing thetemperature in the mold after an initial gradient formation period:gelation proceeds much faster at higher temperature. In a typicalprotocol, the gel gradient is introduced at a temperature of 20° C. andafter a period of approximately 4 minutes, during which thepolymerizable monomers gel into a non-convecting state, the temperaturein the mold is increased to approximately 50° C. by circulation ofheated water through closed channels provided in the backing plate.After removal of the gel from the mold, the temperature is lowered to20° C. by switching the circulation system to a chilled water supply inpreparation for the next cycle.

Once the gel is polymerized, the mold is opened and the IPG gel istransported by manipulation of the Gelbond® support to which it hasbecome covalently attached during polymerization. The form of the gel isdetermined by the form of the mold in which it is cast, the simplestbeing a flat, rectangular strip on the surface of the Gelbond®.

In a further embodiment, the gradient stream of polymerizable monomersis introduced into the mold cavity by means of a passage at the bottomof the cavity, in this case in the sequence light to heavy (opposite tothe order when liquid is deposited at the rising surface of the liquidin the mold). A special valve is used to direct the flow ofpolymerizable liquid either into the mold or to waste, thereby allowingthe contents of the delivery tubing to be purged of polymerizablecomponents after casting of a gel.

Numerous alternative forms of the IPG gel can be produced. In onealternative embodiment, a pin is introduced into the mold before orduring gel polymerization and slowly withdrawn afterwards, leaving acentral hole down the length of the IPG gel. This can be accomplished bya procedure in which the pin is first rotated slowly, to reduce theadhesion of gel to the pin, and subsequently slowly withdrawn along itsaxis through the top of the mold. In another embodiment, a sampleintroduction channel or groove is formed at the exterior surface of theIPG gel by means of a suitably shaped ridge on the interior surface ofthe mold. The groove may be formed so as to be closed at its ends, thusforming a bounded depression, open only at the top. Provided that thegel is held horizontal during the run, i.e. with the groove in ahorizontal plane and with its opening directed upward or to the side,then sample liquid placed in this groove will remain held there bycapillary action, until imbibed by the gel or evaporated.

FIG. 5 shows a series of six alternative cross-sections for IPG gelsformed by various mold cavities, after the strip has been cut from theGelbond® roll. In diagram A, a semi-circular gel 43 with longitudinalhole 45 has been formed on the Gelbond® strip 46 and subsequently filledwith sample. In diagram B, a semi-circular cross-section gel has asurface groove 47 in which sample is held by capillary action, while indiagrams C and D, other cross-sections with broader, flatter surfacegrooves 47 are shown, also holding sample by capillarity. In diagrams Eand F, triangular and rectangular cross-sections without surface groovesare shown. In each case shown, the Gelbond backing material is widerthan the gel itself, giving the strip greater stiffness and providing,particularly in E and F, a further form of cavity in which sample isheld by capillarity: a groove created by the included angle between theside of the gel on one hand and the extended Gelbond substrate on theother.

In practice, the gel mold can be formed from any of a range of materialsthat do not inhibit polymerization of acrylamide, including glass,alumina, machinable ceramic, Ultem®, polysulfone, polystyrene,polycarbonate, polyurethane, acrylic, polyethylene or the like. Forconvenience in machining, and to allow observation of the mold'scontents, a clear plastic such as polysulfone or acrylic is preferred.

Gelbond® Transport

Gelbond® substrate is advanced to the mold on repeated cycles from alarge roll by feed rollers. After casting an IPG gel on the end of theGelbond® (the IPG axis perpendicular to the length of the Gelbond® andparallel to the roll's axis), the strip of Gelbond® on which the gel isformed is cut from the roll using any of a variety of mechanical cuttingmechanisms, including, for example, a rolling disk cutter of the typeused to cut photographic paper, affixed to a vertical motion device. Theresulting Gelbond® strip with IPG gel attached may then be grasped byany of a variety of mechanical or manual means for handling in furtherprocessing steps. In the preferred embodiment, the strip is 1.27 cm wideand approximately 65 cm long (the width of the Gelbond® substrate whenprovided in roll form). The IPG gel is 2 mm wide, 0.75 mm thick and 57cm long (leaving 1.5 cm of the Gelbond® uncovered on either end of thestrip).

Barcode Labeler

Preprinted barcoded labels are mechanically applied to each IPG-carryingstrip on the side opposite the gel for identification purposes, althoughother labeling means known or available may be used.

Robotic Arm

A robotic arm system equipped with two pneumatically-activated pincersgrasps the strip by the two ends to transport it between subsequentprocessing stations. The IPG arm system moves horizontally along atrack, vertically along a linear table mounted on the track, and canrotate 90 degrees in order to pick up the IPG lying in a horizontalposition and carry it in a vertical orientation to subsequent stations.

Sequence of IPG Processing Events

To employ a gel made by the procedure described above as the firstdimension separation of a 2-D electrophoresis procedure, a sequence ofprocessing operations, many of which have been well described in theart, is used to render the get ready for use in a protein separation.These operations include removal of remaining unpolymerized monomers,initiator and catalyst by washing in deionized water; dehydration toremove incorporated water; and finally rehydration in a solutionappropriate as a medium for protein separation. Subsequently, aprotein-containing sample is applied to the gel, and the gel issubjected to a voltage gradient in order to separate the proteins alongthe gel length.

In the preferred embodiment, the IPG gel on its Gelbond® strip isgripped at both ends by the aforementioned movable arm and placed in oneof a plurality of slots containing circulating purified water. Afterapproximately two hours, most soluble materials remaining in the gelhave diffused into the water and are thus removed from the gel.

The strip is then grasped again by the arm (which in the meantime mayhave moved to other positions to carry out other functions) and moved toa slot where it is subjected to a stream of air filtered so as to removeany contaminating particulate material (e.g., using a conventional-HEPAfilter). The gel is substantially dried in approximately 30 minutes.

Next, the arm again grasps the strip and moves it to a slot filled withrehydration solution, a medium typically consisting of 9 mole/literurea, 2% of a non-ionic detergent such as Nonidet P-40 or CHAPS, and 2%wide range, commercially-available ampholytes (e.g., BDH 3-10ampholytes) in water. When samples are to be used whose protein SHgroups have not been alkylated, 1% dithiothreitol is included in therehydration solution as a sulfhydryl reducing agent. In a period ofapproximately two hours, the IPG gel is re-swollen in rehydrationsolution and ready to be used for protein separation. In order toprevent the formation of crystals due to evaporation at the surface ofthe rehydration solution bath, the rehydration solution is covered by alayer of light silicone oil, through which the IPG is inserted.

To carry out a protein separation, a volume of sample protein must beapplied to the gel. In the preferred embodiment, sample protein in asolubilization solution similar in composition to the rehydrationsolution is applied on the surface of the IPG gel along its length. Thisapplication is effected by placing the IPG on a base plate with the gelface up, and depositing a stream of sample liquid onto the IPG gelsurface from a needle held just above that surface, which is movedslowly along the length of the IPG as sample is pumped out. Theresulting thin layer of protein-containing liquid on the IPG gel surfaceremains in place during subsequent manipulations of the gel strip solong as the axis of the gel remains in a horizontal plane (as is thecase during movement using the arm system described). Means are providedfor moving the needle up and down (to allow collection of sample bypiercing the septum of a conventional septum-topped sample vial), andfor moving it along the length of the IPG and farther, to positionswhere a sample vial may be placed and where the needle may be washed.

FIG. 6 shows an apparatus for application of sample protein to anisoelectric focusing gel in accordance with the present invention. AnIPG strip 46 lies horizontally, gel-side up, on a base plate 48. A trailof sample liquid 49 is left on the surface of the IPG gel as needle 50discharges a steady stream of sample while moving along the IPG. Theneedle 50 is moved on carriage 51 through the action of lead screw 52driven by motor 53. Sample flow is controlled by syringe 54 whoseplunger 55 is moved by a block 56 which is in turn moved by a lead screw57 turned by motor 58. Flexible tube 59 connects the syringe and thedelivery needle. The sample is initially taken into the needle 50 andsyringe 54 by raising the needle on its vertical pneumatic motion 60,driving the needle 50 to the left, positioning it over sample vial 61,lowering the needle 50 to pierce the vial's septum top, withdrawing thesample through action of the syringe 54, raising the needle 50 again,moving into position over a gel strip, lowering the needle 50 andcommencing synchronous motion of the syringe 54 and the needle carriage51 to deposit the sample along the IPG surface. The needle 50 is washedbetween applications by positioning it over waste receptacle 62, whereits exterior surface is washed by a jet of water 63.

In another embodiment, where a central hole is produced in the IPG gelduring casting, the sample can be injected or peristaltically drawn intothe channel prior to application of voltage along the gel. The sampleliquid can be retained inside the channel by pinching the ends of thegel to close the channel, by injection of gas bubbles, or by variousother means, including placing a drop of gelling material at both ends.

After sample loading, the gel strip is once again grasped by the arm andmoved to one of a plurality of slots filled with a non-conducting oil(such as silicone oil) and having slotted carbon electrodes at eitherend positioned so as to contact the ends of the IPG gel. The oil may becirculated, cooled to ensure constant running temperature and spargedwith a dry gas so as to eliminate oxygen and dissolved water. Since theresistance of the IPG gel rises during the run, slots maintained at aseries of different voltages are provided, and the arm periodicallymoves the strip from one voltage to a higher voltage as the runprogresses. In the preferred embodiment, a series of 6 voltage stagesare provided, namely 1, 2.5, 5, 10, 20 and 40 kilovolts. The gel ismaintained at each voltage for about 3 hours, except the last, where itrests until a second dimension slab gel is available. A total of 200,000to 300,000 volt-hours may applied to each gel.

Slots such as those used for washing and for subsequent processing andrunning steps generally have clips at either end into which the gelstrip is inserted by the arm, using a downward motion. When the graspingpincers at the ends of the arm release the Gelbond® strip, these clipscontinue to hold the strip extended between them by friction. In thepreferred embodiment, these clips consist of a pair of parallel pinstouching one another and projecting upwards from the floor of the slot.The strip is jammed between these pins during insertion into the slot,spreading them slightly and producing a friction fit. All the slotsexcept the air dryer are contained at the sides and below to yield aliquid-tight vessel suitable for containing the liquid with which theIPG is to be treated at that stage. Slots used for application of highvoltage also contain slotted carbon electrodes.

FIG. 7 shows a cross-section view of an IPG processing slot and the armused to transport IPG strips between slots. A Gelbond® strip 46 carryingattached IPG gel 43 is held at its ends 64 and 65 by a distal arm 70 anda proximal arm 68, each carrying a gripper 66 actuated by a pneumaticcylinder 67. Both arms are mounted on a horizontal bar 69. One of thearms, in this case the distal arm 70, is mounted to a carriage 71capable of moving along bar 69 under the control of belt 72, which inturn is moved by motor and pulley 73. Since the other arm 68 is fixed tothe horizontal bar 69, movement of arm 70 by the motor and pulley in anoutwards direction serves to stretch strip 46, keeping it taut (andtherefore straight) between grippers 66. A vertical motion 74 serves toraise and lower the entire arm and bar assembly, thus allowing insertionof IPG gels into, and removal of gels from, the slots. The verticalmotion is itself carried on motor-driven wheels 75 which engage a track76 to move the arm assembly to positions over a variety of slots.

Movement of the arm assembly downwards (by motion of vertical motion 74)causes gel strip 46 and attached IPG gel 43 to be inserted into aprocessing slot in plate 77. The strip is held at its ends between pairsof pins 78 projecting from the floor of the slot, and is insertedbeneath the surface of liquid 79. This liquid can be circulated over theIPG strip by introducing liquid through inlet 80 and simultaneouslywithdrawing liquid through outlet 81. Excess liquid flows over a dam 82to exit via overflow 83. In slots devoted to the IEF process (wherevoltage is applied across the gel) the ends of the IPG gel 43 contactslotted electrodes 84, which are connected in turn to conducting pins 85that penetrate the bottoms of the run slots in a liquid-tight manner,allowing electrical connection to a power supply on the outside.

During the early stages of a separation run, under an applied electricfield, proteins can migrate through the liquid phase of the appliedsample along a pH gradient initially formed by the action of theampholytes incorporated in the sample. Because the proteins areinitially migrating through liquid, without the retardation associatedwith migration through a gel matrix, they can approach their isoelectricpoints more rapidly than in a system where the entire migration path isthrough IPG gel. However, if proteins remained in this liquid phase atthe end of the run, they could be displaced from their isoelectricpositions by subsequent gel handling steps. Hence, conditions arecontrived so that, as the run progresses, sample-containing liquid isimbibed by the gel, progressively shrinking the channel so that at theend of the run the channel contains a negligible amount of liquid. Thisis achieved by allowing surface water to be slowly removed from theexterior surface of the gel during the run by, for example, immersion ofthe gel in circulated silicone oil that has been dehydrated by spargingwith a dry gas such as argon or nitrogen.

During gel dehydration, and consequent collapse of any liquid filledcentral sample channel, proteins enter the gel at positions near theirrespective isoelectric points. Thus, a mixture of different proteinswill enter the gel at points distributed along the gel length, ratherthan at one site at the edge of a sample well, thereby avoiding theprecipitation often observed when a complex mixture of proteins migratestogether into the gel through a small gel surface area. Excess liquid isremoved through the exterior gel surface, either to a dry gas phase orto a water-extracting, non-aqueous, non-conducting liquid phase such assilicone oil.

SDS Electrophoresis

Slab Gel Casting

In the preferred embodiment, a gel is formed in a computer-controlledmold system whose operation is shown diagrammatically (in cross-section)in FIG. 8. The mold is composed of two halves 86 and 87 which can beforced together to comprise a liquid-tight cavity open at the top. Theform of the mold is such that the gel 89 formed therein has a large,thin planar region at the bottom (within which proteins will beseparated: the “separating gel”)and above the thin planar region asubstantially wider region (the “top gel”)joined to the thin region by ajoining region of gradually increasing width. The function of the topgel is to provide a buffer reservoir as a source of ions during theelectrophoresis separation, and a mechanical support from which theseparating gel hangs during the run and subsequent steps. The joiningregion joins the separating and top gel regions and provides a graduallynarrowing cross-section adapted for the focusing of protein zones usingthe stacking process disclosed in Laemmli (U.K., 1970, Nature 22, 680),in which the joining region is comprised of a stacking gel. In thepreferred embodiment, the separating region has a thickness of about 1mm, the top region has a thickness of about 2 cm, and the joining regiongives rise to a smooth fillet between the separating and top gels. Thevertical height of the separating gel is 30 cm and that of the top gelis 5 cm. All gel regions have the same width, namely 60 cm.

Mixtures of polymerizable gel monomers are introduced into the closedmold by means of three tubes 88, 90 and 95 which can be made to extenddown into the mold cavity from above. The first delivery tube 88 can becaused to extend to the bottom of the mold and is used to introduce aliquid stream that polymerizes to yield the separating gel 89. A seconddelivery tube 90 can be made to extend down inside the upper, widersection of the gel mold, and is used for the introduction of the secondgel phase (the stacking gel 91) and (by means of switching a valve) anequilibration solution used to bathe the IPG applied to the slab gel. Athird delivery tube 95 also can be made to extend into the upper sectionof the gel mold, and is used to introduce the liquid that polymerizesinto the top reservoir gel phase.

A slot form 92 can be lowered into the open top of the mold cavity byvertical movement of the slot form. The mold can be opened by means ofanother movement, whereby one face of the mold pivots along a line nearto and parallel with the bottom horizontal edge of the mold cavity toexpose the gel. The mold cavity contains indentations at either endshaped so as to receive and support the ends of a carbon electrode rod94 and suspend it inside the top gel volume during its polymerization.After polymerization of the gel, electrode rod 94 serves as both anupper electrode required for the electrophoresis separation and amechanical support from which the gel hangs during subsequent handlingand manipulation. A further controlled motion is provided to clamp theelectrode rod to one face of the gel mold, thus ensuring that the gelwill always be recovered in a fixed location after the mold is opened.

FIG. 8 illustrates the sequence of actions of slab gel mold during thecasting operation. In configuration A, a slab gel mold comprised of afixed mold half 86 and a movable mold half 87 is shown in the closedposition. A long delivery tube 88 is extended downwards to the bottom ofthe mold, and the polymerizable mixture which will form the separatinggel is dispensed. The motions of this tube and other delivery tubes arecontrolled by simple vertical electromechanical movements. Inconfiguration B, after the separating gel 89 is polymerized, a secondshorter delivery tube 90 is lowered and a stacking gel phase isdispensed. In configuration C, before the stacking gel 91 polymerizes, aslot form 92 is inserted into the mold to form the sample slot 93. Inconfiguration D, once the stacking gel is polymerized, the slot form iswithdrawn, an electrode rod 94 is inserted into the mold, and a thirddelivery tube 95 is lowered into the mold to dispense a top gel mixture.In configuration E, after the top gel 96 is polymerized, the mold isopened. Once the mold is opened, a completed slab gel 97 hanging fromthe electrode rod 94 is slowly and evenly removed by slab gel handlingarm 98 having an actuated gripper 99. The arm is carried vertically andhorizonally by linear motion components 100 and 101.

FIG. 9a illustrates alternative forms of slab gels. The preferred formof slab gel shown in configuration A comprises three gel phases(separating gel 89, stacking gel 91, and top gel 96), an internalslot-shaped cavity 93 to accommodate the IPG first dimension gel 46, anda rod-shaped electrode 94. In configuration B, the stacking gel phase iseliminated and the internal slot 93 is formed directly in the separatinggel 89. In configuration C, the sample slot 93 extends to the top gelsurface, while two internal electrode rods 94 a and 94 b are used. Inconfiguration D, the sample slot 93 also extends to the upper surface,but the electrode rods 102 a and 102 b are external to the gel andsupport it by interacting with lips 103 on the gel's external surfaces.In configuration E, the IPG gel 46 is applied to an external face of thestacking gel phase rather than being placed in an internal slot,remaining in place as a result of surface tension. In configuration F,the IPG gel 46 is also applied externally, but to the separating gel 89(the stacking gel 91 having been eliminated). In configuration G, thetop phase 96 of a gel configured as in E is rotated counterclockwise byapproximately 160 degrees. By rotating the incorporated electrode rod94, the top gel phase 96 is brought in contact with the separating gel89, bypassing the stacking gel 91 phase and the IPG gel 46, after sampleproteins have entered into the separating phase.

A series of alternative embodiments make use of a gel clamp, instead ofa distinct gel region, to provide an electrode and source of ions. Inconfiguration H (FIG. 9b), a hinged clamp, comprised of halves 104 and105, grasps the top edge of a slab gel and holds it as a result of theclosing force exerted by spring 106. One of the two opposing faces (105)contains an internal cavity 107 and electrode 108, the cavity forming aliquid-tight vessel when the gel is clamped in place thereby coveringopening 109. The gel is prevented from slipping out of the clamp by thepresence of a region of increased gel thickness 110 along the top geledge, in this case including a molded-in rod 111 as a means of handlingthe gel before introduction into the clamp, and secondarily by thepresence of a gritty coating on one or both of the opposing faces of theclamp. Projections 112 above the clamp's axis 113 can be squeezedtogether to open the clamp and release the gel. Axis 113 is connectedelectrically to the liquid vessel's electrode. An IPG gel 46 is appliedon the surface of the slab gel. Once the gel is grasped and the chamber107 is filled with an appropriate volume of electrode buffer, theassembly can be grasped in turn by external means via axis 113, andmanipulated by a robot arm as in the case of the gels with incorporatedelectrode rods (e.g., configuration A). The electrode buffer solutionprovides the source of ions for electrophoresis, using the axis 113 as aconvenient external electrical contact.

In configuration I, a similar clamp is used to grasp a planar slab gelhaving no region of increased gel thickness along the top gel edge. Thegel is prevented from slipping out of the clamp only by the graspingforce and the presence of a gritty coating 114 on one or both opposingfaces of the clamp. In configuration J, the IPG gel is placed within theclamp on a support structure 115, and thereby held against the slab gel.The buffer-containing internal cavity is formed to provide two paths ofcurrent flow 116 and 117 into the slab gel: one above and a smaller onebelow the IPG. This arrangement provides a means for directing theproteins transported from a surface-applied IPG during electrophoresisinto the center plane of the slab gel. Hence, instead of moving alongthe surface of the slab to which they were applied (in the case wherethe IPG is applied to a surface, rather than inside of the slab), theprotein zone is pushed towards the interior of the gel by the flow ofbuffer ions entering through the second path 117. In configuration K,the clamp contains a channel 118 through which buffer can be circulated.One leg of this channel 118 runs along the top edge of the slab gel,where one of the channel's walls is comprised of the gel's surface, andcontains an electrode 108. This channel further communicates throughadditional passages 119 with an external buffer circulation system. Inthis embodiment, buffer is circulated through the clamp during the run,providing a supply of fresh buffer components which, with the electrodemounted in the channel, allow sustained electrophoresis with a minimumvolume of reagents.

In the preferred embodiment, a separating gel (usually a gradientcomposition varying between approximately 18% T acrylamide at the bottomof the gel mold to 11% T acrylamide at the top of the separating gelphase) is introduced through the first delivery tube 88 (FIG. 8A) whileit is extended to the bottom of the mold cavity. This gradient isproduced by a second gradient maker similar in structure to thatdisclosed above to create an IPG gradient, except that larger syringesare used to produce a total separating gel volume of approximately 200ml. After the gel is introduced, the first delivery tube 88 is raisedout of the mold so that its open end lies in a block with vacuumchannels that direct a stream of air across the end of the tube and thusaspirate emerging liquid into a waste container. Multiport valvesassociated with the gradient maker syringes are switched so that thesyringes may be refilled, and so that a supply of pressurized water isconnected with the manifold leading to the delivery tube, thus purgingit of polymerizable components and flushing it with water. Thesetechniques for providing and aspirating delivery wash solvent functionin a manner similar to that described above for IPG gel formation. Theseparating gel is left undisturbed to polymerize for approximately 5minutes.

After initial polymerization, a second gel phase, a stacking gel 91, isformed by extending the second delivery tube 90 into the top of the moldand dispensing approximately 50 ml of polymerizable stacking gel mixturedirectly atop the separating gel. The stacking gel 91 mix is formed bycombining the output of three computer-controlled syringes deliveringstacking gel mix, ammonium persulfate and TEMED. Before this gel phasepolymerizes, the slot form 92 is caused to move down into the top of theslab gel mold. The slot form 92 consists of a thin strip (˜1 mm thick)of plastic mounted so as to present a vertical edge that lies on themold center line which extends to within 1 cm of the separating gel topand within 1 mm of the diverging walls of the mold in the joiningregion. The slot form 92 is approximately 58 cm wide, leaving a 1 cmopen space at either of its ends.

The stacking gel 91 volume is so contrived that the joining region isfilled with stacking gel mixture up to a depth on the slot form ofapproximately 3 mm. Upon polymerization of the stacking gel 91, the slotform 92 thus creates a slot 3 mm deep in the horn-shaped stacking gelcross-section, into which an IPG gel 46 or other protein containingsample may be placed.

After polymerization of the stacking gel 91, the slot form 92 iswithdrawn from the mold, and the arm system used for IPG manipulation isused to place an IPG strip in the slot so formed. Once this arm is againremoved from the mold area, the second delivery tube 90 is once againintroduced into the mold, and a volume of IPG equilibration solution isdispensed through it into the slot occupied by the IPG. Thisequilibration solution (consisting of 10% glycerol, 5 mM DTT, 2% SDS,0.125M Tris HCl pH 6.8 and a trace of bromophenol blue) serves to infuseSDS into the IPG gel 46 and alter its pH to that of the stacking gel 91in preparation for stacking. The second delivery tube 90 is then onceagain removed from the mold.

A second movable arm system then carries a carbon electrode rod 94 (orrods 99) to the mold and positions it within the mold, approximately 1cm from the top of the mold cavity. The electrode rod ends rest inindentations at the ends of the mold cavity, maintaining the rod inposition when released by the arm, which moves away from the mold afterdepositing the rod. The third delivery tube 95 is then introduced intothe mold where it dispenses the third gel phase 96 (the top gel),filling the mold to the top. This top gel phase 96 is produced by aperistaltic pump system combining four components: an acrylamide/bissolution, a buffer solution, ammonium persulfate and TEMED.

The result is a slab gel in three phases, with the IPG first dimensiongel 46 and a carbon electrode rod 94 polymerized inside. Thepolymerizable gel solutions for these three phases are designed topolymerize rapidly, so that the three phases adhere to one another andyield an integral gel whose regions have distinct electrochemicalproperties.

Preferred compositions for the three phases are as follows. Acrylaide™(FMC Corporation) is an alternative gel crosslinker which may be used toincrease gel strength in the stacking gel.

Separating gel Acrylamide 13.00% T bis acrylamide 3.8% C Tris HCl pH 8.60.375 M Stacking gel Acrylamide 8.00% T Tris HCl pH 7.0 0.375 MAcrylaide 2% 3.2% C SDS 0.2% Top electrode gel Acrylamide 13.00% T Trisbase 0.048 M Glycine 0.4 M SDS 0.20%

After the gel is made, the mold is opened by moving apart the moldhalves 86 and 87 and leaving the gel on the movable, now nearlyhorizontal, mold half 87. A second computer-controlled arm system,equipped with two graspers or pincers 99 designed to engage the oppositeends of the electrode rod 94, is moved into position to seize theelectrode rod 94 and then lift the gel upward and out of the mold.Gravity causes the gel to hang downwards from the bar.

Slab Gel Electrophoresis

The arm is then moved laterally into position over an empty slot in aslab gel running tank and slowly lowers the slab gel into the slot. FIG.10 illustrates a slab gel running tank in accordance with the presentinvention, wherein a slab gel 97 is suspended vertically in silicone oilduring the second dimension electrophoresis run. The slab 97 issuspended by electrode rod 94 which rests on electrical bus bars 120(one at either end of the gel), with the slab gel 97 inserted into avertical slot through which cooled silicone oil is circulated. The oilcirculation path is so contrived as to cause laminar flow of a curtainof oil downwards along both surfaces of the slab gel, thereby removingjoule heat generated during electrophoresis. The oil is recovered at thebottom of the slots and recirculates through an external pump and heatexchanger, and thereafter is reintroduced into the top of the slot in aclosed-loop system. This curtain-like flow of oil serves to prevent theslab gel 97 from touching the walls of the slot, and insulates it fromelectrical contact along its length. Oil enters the tank throughmanifold 121, is distributed to supply plenums 122, expelled throughholes 123 into the gel slot, and flows down the slot on either side ofthe separating gel 89, to be sucked out through return manifold 124 viareturn plenums 125 and return holes 126.

At the bottom of the tank, below the level of the bottom of the slots, alower electrically-conductive aqueous phase 127 (denser than thesilicone oil) is positioned so that it just contacts the bottom edge ofthe slab gel 97. Current passes from the electrode bar or bars embeddedin the top gel 96 through the stacking gel 91 and separating gel 89 tothe lower aqueous phase and lower electrode 128, thus completing thecircuit required for an electrophoretic separation. The shield 129 isprovided over the lower electrode 128 to funnel the bubbles generatedthere to one side and up a separate pipe, thus preventing their risingthrough the aqueous phase and then the silicone oil phase, and causingmixing of the two phases.

At a voltage of 600 volts and a current of 1 amp, the separation ofproteins in the separating gel 97 can be effected in approximately 4 to5 hours. Once the separation is complete, the aforementioned slab gelarm system is used to grasp the ends of the electrode bar 94, raise thegel out of the running slot and move the gel into position over thefirst of several tanks containing solutions required to visualize theseparated proteins by staining.

Slab gels and electrophoresis methods of the type disclosed can be usedfor separation of samples other than proteins contained in IPG gels. Inparticular, the inclusion of multiple sample wells in place of thesingle slot provided for an IPG allows use of such gels to separateprotein or nucleic acid components of numerous liquid samples.

Slab Gel Staining

Several stain protocols can be executed including, among many others,staining with Coomassie Brilliant blue, ammoniacal silver, silvernitrate, and fluorescent stains such as SYPRO red and orange. Thefollowing example exemplifies the method applied to any stain. The gelis moved between subsequent tanks, by the arm under computer control, sothat the precise time of movement from one solution to the next can becontrolled, and can be held generally constant from gel to gel.

In a first tank, the gel is immersed up to the stacking gel in asolution of 30% ethanol, 2% phosphoric acid and 68% water for a periodof two hours, to fix the proteins in place and remove most of the SDS,Tris and glycine in the gel. Following this fixation step, the gel ismoved, through use of the arm, to a tank of 28% methanol, 14% ammoniumsulfate, 2% phosphoric acid in water, where it is incubated for twohours. Next, the gel is moved to a tank of the same composition with theaddition of powdered Coomassie Blue G250 dye, the whole liquid volumebeing continually circulated or agitated in the tank. Here the dyepermeates the gel, binding to resolved protein spots. Finally, the gelis removed from this tank and transported by the arm to a scanningstation.

FIG. 11A illustrates slab gel staining tanks with a slab carrier arm. Inorder to expose slab gels 97 to staining solutions, the gels aresuspended in staining tanks 130, where they are supported by theembedded electrode rods 94 whose ends sit on projecting supports 131.The tank 130 is filled with stain solution 132, which can be removedfrom the tank by opening exit valve 133. The tank 130 can be refilled byclosing valve 133 and then opening input valve 134 and activating pump135 to deliver solution 132 from reservoir 136. Solutions in the tankcan be agitated when required by a variety of means well known in thephotographic processing industry, including bursts of inert gas (such asnitrogen or argon) introduced at the bottom of the tank, or by smallmechanical motions of the suspended gels caused by cyclic movement ofthe gel supports 131. Gels 97 are moved from tank to tank by means ofarm 98 having pneumatically controlled grippers 99 which seize the endsof electrode rod 94. The arm 98 is raised and lowered by verticalmovement 100 which in turn rides on lateral movement 101, all undercomputer control.

FIGS. 11B and 11C show alternative embodiments allowing gels withoutincorporated electrode rods to be similarly processed. In B, a slab gel89 is contained inside a holder whose two halves 137 and 138 areconnected by hinge 139 at the top edge and held together by magnets 140at the bottom edge. Each half of the rectangular holder has a largecutout and is shaped like a picture frame. One surface of each half iscovered with a taut mesh 141, resulting in a narrow gel cavity withlarge-area porous walls. A slab gel placed in such a holder is thusexposed to any solution into which the holder is immersed, and can beprocessed through a series of tanks using a robot arm to graspprojecting pins 142. In C, an alternative slab gel holder makes use of aclamp hinged at 142, held together by magnets 143 and having itsinternal faces 144 coated with a gritty coating, to grasp a slab gel fortransportation and processing. Projections 145 may be squeezed togetherto open the clamp, releasing the gel.

Scanning

In order to obtain quantitative data on the abundance of resolvedproteins, the gel is scanned to yield a digitized image. FIG. 12 shows agel 97 being gently laid down on a horizontal or tilted illuminatingtable 146 prior to scanning, grasped as before by the electrode rod 94embedded in its top phase 96. To do this, the robotic arm 98 executes acoordinated vertical and horizontal motion so that the gel is laid downsmoothly without tension. An overhead digital camera 147, such as a CCDdigitizer, may then be used to acquire an image of the gel 97 and itsstained protein spots in absorbance mode. In order to allow scanning ofa large area gel at high resolution, a camera covering, for example,1024×1024 pixels can be moved to a series of locations by orthogonallinear motions 148 and 149, generating a series of scans that can becombined to yield a larger image. Alternative scanning and illuminationmodes may be provided for measuring fluorescence or light scattering, insituations where the proteins have been stained with a fluorescent or aparticulate dye, respectively. In the preferred embodiment, fluorescenceexcitation illumination is delivered to the gel in the plane of the gelwhile it lies in a horizontal cavity defined by walls 151 and filledwith a liquid 152, such as water, having a refractive index similar tothe gel. Light is piped into the cavity by an optical fiber light pipe153, one of whose ends pierces the walls 151, the other end beingilluminated by light produced by light source 155 filtered by interposedoptical filter 154. In fluorescence mode, light emitted by fluorescentmoieties in the gel is detected by the digitizer after passage through asecond optical filter 150 which passes the dye's emission wavelengthwhile blocking the excitation light. The approach described makes use ofthe fact that the exciting light is trapped by internal reflections inthe gel/water plane, thus improving its availability to exciteprotein-bound fluorescent dye molecules and diminishing the amount ofexciting light that escapes normal to the gel plane to impinge on thedetector. A similar optical system, but without a requirement forexcitation and emission filters, can be used to detect light scatteringby particles generated either on the protein spots (for example by thesilver stain) or around the spots (leaving the proteins negativelystained, as occurs with the copper stain).

Using the automated staining system described, multiple stain and scancycles can be sequentially applied to the same gel. By staining firstwith a relatively low sensitivity stain such as Coomassie Blue andscanning, and then staining with a relatively sensitive stain such asthe silver stain and scanning once again, it is possible to obtainquantitative protein abundance measurement over a wider dynamic rangethan can be afforded by any single conventional stain.

Multiple sequential scans of the same gel may be used to increase theprecision and dynamic range of non-equilibrium stains such as the silverstain. In such stains, the development process reveals first theintensely staining spots (in general the more abundant proteins), thenthose of moderate staining intensity, and finally those of low stainingintensity (typically low abundance proteins), at which point theintensely staining spots are over stained, being saturated in stainabsorbance and appearing increased in size. By scanning the gel two ormore times during development, quantitation of spots can be based onmeasurements of parameters other than simple optical density. The mostuseful of such parameters include maximum rate of change of absorbance(effectively the maximum slope observed in a plot of optical densityversus time) and time of onset of development (the time after thebeginning, of development at which a given increment of optical densityis observed), both of which can be calculated for each pixel in thescanned gel image through use of multiple scans yielding optical density(or transmittance) as a function of time during the development of thegel. Alternatively, sophisticated curve-fitting algorithms can be usedto devise functions of absorbance as a function of time that yield, foreach pixel, a derived parameter well-correlated with known differencesin abundance.

Multiple scans of the same gel can also be used to compare proteinsamples, provided that the proteins of each sample are labeled prior toelectrophoresis with a dye or other substituent that can be detectedseparately from other such labels. Multiple samples labeled with aseries of different fluorescent dyes having distinct emissionwavelengths, for example, can be mixed and co-electrophoresed. By usingappropriate optical filters to detect these dyes (and thus the proteinsto which they are bound) separately, the protein content of each samplecan be measured separately from the protein contents of other samplesapplied to the same gel. When used in a 2-D procedure that includesisoelectric focusing, such labels must be attached to the protein insuch a way that the protein's net pI is unaffected: if, for example, thelabel is attached by reaction with a lysine primary amino group, thenthe label must have a net charge of +1 to compensate for the singlepositive charge of the primary amino group lost when the lysine isderivatized. While this approach increases the information output ofeach separation (by multiplexing samples), it also makes possible asubstantial increase in net resolution available for the comparison ofsamples. This comes about because the different label distributionsobserved in a small gel region (a protein spot in a 2-D electrophoresispattern) can be compared with great sensitivity by mathematicaltechniques to determine whether the shape and location of a spot in onelabel channel is precisely the same as the shape and location of a spotin another label channel (both labels being detected on the same gelwhere they reveal the proteins of two different samples). Spotpositional differences detectable by this approach (using for example acorrelation coefficient to determine whether the spot profiles in twochannels are the same or different) can be on the order of 0.1 mm, farless than the 0.5-2.0 mm position difference typically required tocharacterize protein spots as being different when two different gelsare compared, or when two samples are co-electrophoresed on one gel andstained with a single stain. When applied to both dimensions of a 2-Dprocedure, this method of comparing potentially co-electrophoresingproteins can result in an effective 100-fold increase in net gelresolution (the product of an approximate 10-fold resolution increase ineach dimension). Such an approach is of particular value in comparingvery different protein patterns (for example different tissues), whereit is likely that different proteins with similar 2-D gel positions maybe encountered.

Spot Excision

Protein spots can be excised from the gel under computer control oncetheir positions are established by the aforementioned scanning. FIG. 13shows a mechanical cutter comprised of a block 156 in whose lower part athin-wall tube 157 is mounted vertically to act as a spot-cutting punch.The block and all its components are mounted on a movable, computercontrolled X-Y frame, suspended just above and co-planar with the gel,such that the cutter 157 can be positioned over any spot to be excisedfrom the gel. A plunger 158 is arranged so as to moved vertically withinthe punch. The plunger extends through a hollow cavity 160 in the blockand exits through a second hole by means of channel containing an O-ringseal 159. The plunger is moved vertically by an actuator 161, and theblock is moved vertically by a second actuator 162 having less force,and thus capable of being overridden by actuation of the plungeractuator. The gel to be cut 97 lies horizontally on a flat plat 163,which can be identical to the scanning platform/lightbox 146. Inoperation, the cutter performs a series of steps as shown in the figure.In configuration A, the block is positioned over the spot to be cut. Inconfiguration B, the plunger actuator is pressed down, forcing theplunger to protrude through the cutting tube 157 into close proximitywith the gel surface and further forcing the block partially downthrough interaction of collar 164 on the plunger with the block. Inconfiguration C, actuator 162 is forced down, forcing the cutter throughthe gel and into contact with the supporting plate 163. In configurationD, the plunger actuator 161 is pulled upwards, moving the block up byinteraction of collar 164 with the block and simultaneously generatingsuction in the cutter tube so as to ensure that the cut gel plug 165 islifted away from the gel by the upwards motion. In configuration E, thecutter has been repositioned over a collection vessel 166, and theplunger forced down to expel the gel plug into the vessel. Inconfiguration F, with both actuators in the up position, a stream ofwash liquid is introduced through hole 167 in the block 156 so as toexpel any contaminating particulate gel material remaining in the punchinto a waste receptacle 168. Under computer control, the spot cuttingmechanism can excise hundreds of spots from a single 2-D proteinseparation, depositing them in 96-well plates or other vessels forsubsequent analysis by other means such as mass spectrometry. In thepreferred embodiment, the spot cutter mechanism is incorporated into thegel scanning system, thus allowing the gel to be cut in an automatedfashion immediately following computer analysis of the gel imageobtained from the scanner.

System Scheduling Algorithms

Operating as a continuous production line, the automated 2-D gel systemdescribed must allow flexible scheduling of each component action in themulti-step process required to make and run each gel. If every gel wererun using the same protocol, it would be possible to design a completelysynchronous scheduling system in which each action recurred at preciselydefined intervals. However, such a system is inherently inflexible andwould not allow running successive gels with different parameters (e.g.,different IPG pH gradient, focusing volt-hours, or time in a stainsolution). In addition, any temporary halt required in such asynchronous system, due for example to an equipment breakdown, wouldcause variable and unforeseen consequences at different stages of theprocess.

Hence in the preferred embodiment, a non-synchronous schedulingalgorithm is used in which a series of steps is laid out for the firstsample to be run, and these are entered into a database of actionsrequired, each step associated with a relative or absolute time at whichit should be executed. Then a second series of steps is laid out for thesecond sample to be run, and these are entered into the databaseincluding a start delay calculated so as to prevent any action requiredfor the second gel from being interfered with by any action required forthe preceding (first) gel. Additional gels are added in order by thesame procedure, ensuring in each case that the actions required for agel do not interfere with those required for previously entered gels.Actions to be entered include casting an IPG gel, transporting an IPGfrom the caster to a wash slot, transporting an IPG from a wash to adrying slot, casting a slab gel, moving a slab gel from mold to runningslot, moving a slab gel from a running slot to a stain slot, etc.Database entries take account of the time required to execute suchactions, e.g., the time to move a gel from one station to another or toempty and refill a stain tank. The sequence of operations required toeffect the processing of a series of gels, including interleaving ofactions on different gels, is readily obtained by retrieving from thedatabase a series of steps sorted by time of scheduled execution. Makinguse of the ability of database software to sustain multiple independentqueries, different software modules controlling specific parts of thehardware system may retrieve a subset of actions (in scheduled timeorder) appropriate to them.

The automated system is then operated under the control of one or morecomputer programs which function by examining the database of scheduledactions, selecting from the database those actions appropriate to thehardware components being controlled by that program, and executing themat the time specified in the appropriate database record. Hence, asingle IPG manipulation arm will be caused to transport IPG gels atdifferent stages of the process between the required slots and stations,actions on different gels thus being interleaved in a flexible manner.Since each gel is separately scheduled at the outset, it can have adifferent protocol or different parameters than the preceding orsucceeding gel, without limitation.

Data Reduction

Scanned images of 2D protein patterns are subjected to an automatedimage analysis procedure using a batch process computer software (e.g.,Kepler® software system). This software subtracts image background,detects and quantitates spots, and matches spot patterns to master 2Dpatterns to establish spot identities. The final data for a 2-D gel, aseries of records describing position and abundance for each spot, arethen inserted as records in a computerized relational database.

Other Uses and Embodiments

The methods disclosed herein can be used for a series of alternativeanalytical applications including the analysis of DNA and RNA, as wellas peptides. Either the automated IPG or slab gel system can be used forhigh-throughput one-dimensional analyses of relevant biomolecules aswell as for 2-D.

It will be appreciated that the methods and structures of the presentinvention can be incorporated in the form of a variety of embodiments,only a few of which are described herein. It will be apparent to theartisan that other embodiments exist that do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

What is claimed is:
 1. A method of excising a plurality of small regionsof an electrophoresis gel comprising resolved proteins, said methodcomprising steps of: a) staining said gel; b) scanning said gel to yielda digitized image; c) inputing said image into an image processor foridentifying a plurality of protein-containing regions in said gel; d)excising said protein-containing regions from said gel by means of acomputer-controlled movable punch device on the basis of coordinatessupplied by said image processor, wherein said punch device comprises atube-like cutter and a central piston within said cutter, said cutterand said piston being moved coaxially by a computer-controlled means tocut, retrieve and expel a plug of said gel; and e) depositing saidprotein-containing regions in a plurality of vessels.
 2. The method asset forth in claim 1, wherein in said excising step, said tube-likecutter applies suction to said plug for picking up said plug.
 3. Themethod as set forth in claim 1, further comprises a moving step whereinafter said staining step, said gel is moved onto a generally horizontalsurface for said scanning step.
 4. The method as set forth in claim 3,wherein said gel is laid down smoothly without tension on said generallyhorizontal surface.
 5. The method as set forth in claim 4, wherein saidgel remains on said generally horizontal surface for said excising step.6. The method as set forth in claim 1, further comprising: a secondstaining step after said scanning step; and a second scanning step aftersaid second staining step.
 7. A method of excising a plurality of smallregions of an electrophoresis gel comprising resolved proteins, saidmethod comprising steps of: a) staining said gel; b) scanning said gelto yield a digitized image using a gel scanning system; c) identifyinglocations of a plurality of protein-containing regions on said gel usingan image processor; d) excising said protein-containing regions fromsaid gel by means of a spot cutter mechanism comprising a punch device,said spot cutter mechanism being incorporated into said gel scanningsystem on the basis of coordinates supplied by said image processor, andwherein said punch device comprises a tube-like cutter and a centralpiston within said cutter, said cutter and said piston being movedcoaxially by a computer-controlled means to cut and expel a plug of saidgel; and e) depositing said protein-containing regions in a plurality ofvessels.
 8. The method as set forth in claim 7, wherein in said excisingstep, said tube-like cutter applies suction to said plug for picking upsaid plug.
 9. The method as set forth in claim 7, further comprising amoving step wherein after said staining step, said gel is moved onto agenerally horizontal surface for said scanning step.
 10. The method asset forth in claim 9, wherein said gel is laid down smoothly withouttension on said generally horizontal surface.
 11. The method as setforth in claim 10, wherein said gel remains on said generally horizontalsurface for said excising step.
 12. The method as set forth in claim 7,further comprising: a second staining step after said scanning step; anda second scanning step after said second staining step.
 13. A spotcutter adapted for electrophoresis analysis, comprising: a computercapable of receiving coordinates of spots in an electrophoresis gel; asupport plate adapted to support said electrophoresis gel; a punch blockincluding a cutting tube of a predetermined size; an actuator connectedto said punch block and capable of moving said punch block verticallyunder control of said computer; a plunger coaxial with said cuttingtube; and a plunger actuator capable of vertically moving said plungerwithin said cutting tube; wherein said computer is capable ofhorizontally positioning said cutting tube in relation to a spot to beexcised from said electrophoresis gel, moving said plunger downward intocontact with said electrophoresis gel, moving said punch block downwarduntil said cutting tube cuts through said electrophoresis gel, movingsaid plunger upward and away from a gel plug contained within saidcutting tube, moving said punch block upward and away from saidelectrophoresis gel, positioning said punch block over a collectionvessel, and moving said plunger downward in said cutting tube to pushsaid gel plug out of said cutting tube.
 14. The spot cutter of claim 13,wherein said support plate is capable of being moved horizontally undercontrol of said computer.
 15. The spot cutter of claim 13, wherein saidsupport plate is capable of being moved horizontally in two dimensionsunder control of said computer.
 16. The spot cutter of claim 13, whereinsaid punch block is capable of being moved horizontally under control ofsaid computer.
 17. The spot cutter of claim 13, wherein said punch blockis capable of being moved horizontally in two dimensions under controlof said computer.
 18. The spot cutter of claim 13, wherein said punchblock moves downward under power of said actuator device.
 19. The spotcutter of claim 13, wherein said punch block moves downward due togravity.
 20. The spot cutter of claim 13, wherein said plunger issubstantially equal in size to an inner dimension of said cutting tube.21. The spot cutter of claim 13, wherein said spot cutter furtherincludes a horizontal drive device connected to said punch block andcapable of moving said punch block horizontally under control of saidcomputer.
 22. The spot cutter of claim 13, wherein said spot cutterfurther includes a washing passage capable of providing a wash liquidthrough said cutting tube.
 23. The spot cutter of claim 13, wherein saidplunger actuator has less force that said actuator connected to saidpunch block.
 24. A computer-implemented method of cutting a spot from anelectrophoresis gel, comprising the steps of: horizontally positioning apunch block and associated cutting tube in relation to said spot to beexcised from said electrophoresis gel; moving a plunger downward intocontact with said electrophoresis gel, with said plunger being coaxialwith said cutting tube; moving said punch block and associated cuttingtube downward until said cutting tube cuts through said electrophoresisgel; moving said plunger upward and away from a gel plug containedwithin said cutting tube; moving said punch block and associated cuttingtube upward and away from said electrophoresis gel; positioning saidpunch block and associated cutting tube over a collection vessel; andmoving said plunger downward in said cutting tube to push said gel plugout of said cutting tube.
 25. The method of claim 24, wherein a computerreceives data containing spot locations on said electrophoresis gel. 26.The method of claim 24, wherein said method further includes the step ofpassing a wash liquid through said cutting tube after said gel plug ispushed out.
 27. The method of claim 24, wherein said gel plug is placedwithin a unique collection vessel, with said unique collection vesselbeing recorded by said processor.
 28. The method of claim 24, whereinsaid punch block is capable of being moved horizontally under control ofsaid computer.
 29. The method of claim 24, wherein said punch block iscapable of being moved horizontally in two dimensions under control ofsaid computer.
 30. The method of claim 24, wherein said punch blockmoves downward under power of said vertical actuator device.
 31. Themethod of claim 24, wherein said punch block moves downward due togravity.
 32. The method of claim 24, wherein said plunger issubstantially equal in size to an inner dimension of said cutting tube.33. The method of claim 24, wherein a horizontal drive device connectedto said punch block moves said punch block horizontally under control ofsaid computer.